Chromosome ends are specialized nucleoprotein structures called telomeres, which are essential for stable chromosome maintenance. Loss of telomere repeats, observed in most normal somatic cells studied to date, has been proposed as the limiting factor in their replicative life-span. In tumor-derived cell lines, telomeres are maintained by the ribonucleoprotein enzyme telomerase or by other, poorly understood mechanisms. It is proposed to investigate the role of individual telomeres and telomerase in both the replicative senescence and immortalization of human fibroblasts and lymphocytes using novel quantitative fluorescence in situ hybridization (FISH) procedures developed by the applicant. In addition, modified FISH procedures will be explored to measure the replicative history and potential of lymphocytes by flow cytometry. For this purpose, lymphocyte (sub)populations from normal individuals, patients after bone marrow transplantation and patients infected with HIV will be analyzed before and after in vitro expansion. The specific aims of this project are: 1. To study the telomere length on individual chromosomes during propagation and senescence of human fibroblasts and lymphocyte clones. 2. To study telomeres on individual chromosomes in immortal cells in relation to telomerase expression. 3. To develop and refine methods to measure telomere repeats in cells and chromosomes. Taken together, the proposed research will delineate the role of individual telomeres in the replicative senescence and immortalization of human cells and explore the use of telomere length measurements as a tool to assess the replicative history and potential of cells.